CN102234336A - Fucoidan-galactosan sulfate, extracting, separating, and purifying method thereof, and application thereof - Google Patents
Fucoidan-galactosan sulfate, extracting, separating, and purifying method thereof, and application thereof Download PDFInfo
- Publication number
- CN102234336A CN102234336A CN 201010157935 CN201010157935A CN102234336A CN 102234336 A CN102234336 A CN 102234336A CN 201010157935 CN201010157935 CN 201010157935 CN 201010157935 A CN201010157935 A CN 201010157935A CN 102234336 A CN102234336 A CN 102234336A
- Authority
- CN
- China
- Prior art keywords
- fucoidan
- falactosan
- sulfuric ester
- fucose
- fuc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a fucoidan-galactosan sulfate with a novel structure. Fucoidan-galactosan sulfate provided by the present invention is a sulfated polysaccharide composed of fucose and galactose. According to the chemical constitution of the sugar chain, a fundamental chain is formed by -4-alpha-L-Fuc-(1,3)-alpha-L-Fuc-(1,3)-alpha-L-Fuc-(1,3)-alpha-L-Fuc-(1-residue. Every 4 sugar residues comprise a branched chain, which is composed of alpha-L-Fuc(1- or beta-D-Gla-(1,6)-beta-D-Gla-(1-. Sulfated group is connected to the 2 or 4 site of fucose, or on the 3 or 4 site of galactose. Meanwhile, the invention discloses a preparation method and an application of fucoidan-galactosan sulfate. Fucoidan-galactosan sulfate provided by the present invention can be exploited into medicines for treating kidney diseases.
Description
Technical field
The present invention relates to biochemical field, is a kind of fucoidan-falactosan sulfuric ester and separation purification method and application specifically.
Background technology
Fucoidin has another name called algal polysaccharide sulfate, English name fucan or fucoidan, be to be present in the sulfated polysaccharides that the big class of one in ocean brown alga or the marine invertebrate is made up of Fucose, have multiple biological activitys such as anticoagulation, antitumor, antithrombotic, anti-inflammatory.People have understanding comparatively clearly to the composition of fucoidan-falactosan now, and it is a class chemical constitution and the very complicated polysaccharide of structure.
The fucoidin chemical structure is very complicated, and its structure of the fucoidin that is separated in the different brown algas has very big-difference.Up to the present, maximum to the structural research of the fucoidin that derives from black wrack (Fucus vesiculosus) and bladder wrack (Ascophyllum nodosum), the black wrack fucoidin mainly connects with α (1 → 3) glycosidic link, and sulfation mainly occurs in C
4The position.The multinomial research of bladder wrack fucoidin all shown wherein have a large amount of α (1 → 3) and α (1 → 4) glycosidic link.Also have the structure of several brown alga fucoidin to be in the news in addition.Kelp (Ecklonia kurome) fucoidin is mainly α (1 → 3) and connects, and sulfation is at C
4The position.The fucoidin main chain that derives from tap algae (Cladosiphonokamuranus) and rope algae (Chorda filum) is the Fucose of α (1 → 3), and sulfation is at C
4The position, and the two all has a spot of 2-O-acetylize.
Black wrack, bladder wrack fucoidin structure
Kelp fucoidin structure
Rope algae fucoidin structure
In fucoidin, also there is the more complicated sulfuric acid mixed polysaccharide of chemical constitution and structure in the part brown alga.Along with the kind difference of algae contains other compositions such as semi-lactosi, wood sugar, glucuronic acid respectively.But, be this saccharoidal research bottleneck for the parsing and the activity research of the chemical structure of such sulfuric acid mixed polysaccharide always.
Summary of the invention
The purpose of this invention is to provide a kind of from the brown alga of ocean isolating fucoidan-falactosan sulfuric ester.It is characterized in that: it mainly contains Fucose and semi-lactosi; (the 1-residue forms main chain to the sugar chain chemical structure with-4-α-L-Fuc-(1 → 3)-α-L-Fuc-(1 → 3)-α-L-Fuc-(1 → 3)-α-L-Fuc-, average per 4 saccharide residues contain a side chain, and (1-or β-D-Gla-(1 → 6)-(1-forms β-D-Gla-side chain by α-L-Fuc; Sulfate is connected on 2,4 of Fucose or semi-lactosi 3,4.
Another object of the present invention provides the preparation method of this fucoidan-falactosan sulfuric ester.
Another object of the present invention provides the application of fucoidan-falactosan sulfuric ester in treatment renal failure, nephrotic syndrome and medicine for treating diabetic nephropathy.
The thick algal polysaccharide that extracts from brown alga through DEAE-Sepharose-CL-6B gel chromatography column purification, obtains the higher fucoidan-falactosan sulfuric ester of a kind of purity.Through the HPLC spectrum analysis, as Fig. 1, the monose of fucoidan-falactosan sulfuric ester composition mainly contains Fucose and semi-lactosi.
For determining the sulphating position, the method that adopts dimethylsulfoxide solvent to separate obtains the fucoidan-falactosan behind the desulfurization acidic group, the one dimension that compares fucoidan-falactosan sulfuric ester and desulfurization fucoidan-falactosan sulfuric ester, two dimension NMR spectrogram (table 1,2 and Fig. 4-8), in conjunction with methylation analysis result (table 3) and data in literature, the primary structure of releasing the fucoidan-falactosan sulfuric ester be with-4-α-L-Fuc-(1 → 3)-α-L-Fuc-(1 → 3)--(the 1-residue forms main chain to 3-α-L-Fuc-(1 → 3)-α-L-Fuc-, average per 4 saccharide residues contain a side chain, and (1-or β-D-Gla-(1 → 6)-(1-forms β-D-Gla-side chain by α-L-Fuc.Sulfate radical is connected on 2,4 of Fucose or semi-lactosi 3,4.Its repeating unit can be expressed as follows:
Table 1 desulfurization fucoidan-falactosan sulfuric ester
1H-NMR and
13The C-NMR data
Table 2 fucoidan-falactosan sulfuric ester
1H-NMR and
13The C-NMR data
The part methyl data of table 3 fucoidan-falactosan sulfuric ester and desulfurization fucoidan-falactosan sulfuric ester
The separation purification method of above-mentioned fucoidan-falactosan sulfuric ester, its step is as follows:
(1) brown alga is removed silt, add water, 60-120 ℃ of heating extracted 1-4 time; United extraction liquid, suction filtration, concentrating under reduced pressure is used ethanol sedimentation, the dry crude extract that gets; Described temperature is 60-120 ℃; The described amount that adds water is a 10-40 times of frond amount; Described suction filtration helps filter with diatomite; Described alcoholic acid final volume concentration is 50-80%;
(2) crude extract is added water and redissolve (making its concentration is 1-1.5%), add magnesium chloride and ethanolic soln, the centrifugal precipitation of removing is collected supernatant liquor; Supernatant liquor adds ethanol sedimentation again through concentrating under reduced pressure, and alcoholic acid final volume concentration is 50-80%; The final concentration of wherein said magnesium chloride in the redissolution thing is 0.05mol/L, and ethanol final weight concentration is 20%.
(3) will precipitate water-solublely, and be the dialysis of 3500Da dialysis tubing with molecular weight cut-off, and after dialysis finishes solution decompression in the bag be concentrated, lyophilize gets the algal polysaccharide of purifying.
(4) algal polysaccharide that (3) are obtained water-soluble (making its concentration is 1-3%), with DEAE-Sepharose-CL-6B is the carrier column chromatography, use 0.1mol/L NaCl, 0.5mol/LNaCl, 1.0mol/L NaCl, 1.5mol/L NaCl and 2.0mol/L NaCl solution linear gradient elution successively, collect each elutriant, respectively dialysis, freeze-drying; Wherein the white sample by 1.0mol/L NaCl wash-out gained is the fucoidan-falactosan sulfuric ester.
Beneficial effect of the present invention: a kind of a kind of fucoidan-falactosan sulfuric ester that provides is provided the present invention's extraction separation purifying from brown alga, this polysaccharide has better curative effect in treatment renal failure and diabetic nephropathy, in preparation is used for the treatment of the medicine of chronic renal failure, important use is arranged, its market outlook are wide, are worth high.
Description of drawings
The monose of Fig. 1 fucoidan-falactosan sulfuric ester is measured HPLC spectrogram (figure A is a standard monose, and figure B is the fucoidan-falactosan sulfuric ester);
The infrared spectrum of Fig. 2 fucoidan-falactosan sulfuric ester (A) and desulfurization fucoidan-falactosan sulfuric ester (B);
Infrared spectrum after Fig. 3 fucoidan-falactosan sulfuric ester (A) and desulfurization fucoidan-falactosan sulfuric ester (B) methylate;
Fig. 4 fucoidan-falactosan sulfuric ester
1H NMR and desulfurization fucoidan-falactosan sulfuric ester are at the amplification spectrogram of 4.4-5.4ppm;
Fig. 5 fucoidan-falactosan sulfuric ester
13C NMR and desulfurization fucoidan-falactosan sulfuric ester are at the amplification spectrogram of 93-105ppm;
The COSY spectrogram (left side) of Fig. 6 fucoidan-falactosan sulfuric ester and desulfurization fucoidan-falactosan sulfuric ester are at the amplification spectrogram (right side) of 4.4-5.4ppm;
The hsqc spectrum figure (left side) and the fucoidan-falactosan sulfuric ester of Fig. 7 desulfurization fucoidan-falactosan sulfuric ester partly amplify spectrogram (right side);
The HMBC spectrogram of Fig. 8 desulfurization fucoidan-falactosan sulfuric ester and part are amplified spectrogram.
Embodiment
Below by embodiment the present invention is further specified.Here want to be pointed out that, below embodiment only be used for illustrating the present invention, those skilled in the art are understanding under the prerequisite of spirit of the present invention, can carry out corresponding conversion to the present invention according to the prior art and the knowledge in present technique field, these technical schemes all fall within the scope of the present invention.
Embodiment 1: the preparation of fucoidan-falactosan sulfuric ester
(1) the 5kg sea-tangle is removed silt, in pressure kettle, extracted 3 hours, controlled temperature 100-105 ℃, extract 2 times with the water of 20 times of sea-tangle weight.Remove frond, merge No. 2 times extracting solution, use the diatomite suction filtration, filtrate concentrates, and adds the dehydrated alcohol precipitation of 4 times of volumes of concentrated solution, filter Crude polysaccharides, the polysaccharide crude yield is: 4.84%.
(2) be 1.5% solution with the water-soluble mass concentration that is made into of Crude polysaccharides, add 2mol/L MgCl
2Make MgCl
2Whole mass concentration is 0.05mol/L, adds dehydrated alcohol simultaneously, and making the whole weight concentration of ethanol is 20%, stir and generate precipitation, the centrifugal precipitation of removing is got supernatant liquor and is added 95% ethanol, making the whole weight concentration of ethanol is 70-75%, stirs and generates precipitation, centrifugal collecting precipitation, with the water-soluble aqueous solution that is made into mass concentration 2% of precipitation, pack in the dialysis tubing,, concentrate solution in the dialysis tubing with 3500Da dialysis tubing dialysis 2 days, freeze-drying gets algal polysaccharide, and yield is 2.71%.
(3) it is water-soluble to get the algal polysaccharide that above-mentioned freeze-drying obtains, be made into concentration and be 2.5% the aqueous solution, last sample is to being the carrier column chromatography with DEAE-Sepharose-CL-6B, use 0.1mol/L NaCl, 0.5mol/L NaCl, 1.0mol/L NaCl, 1.5mol/L NaCl and 2.0mol/L NaCl eluant solution successively, collect each elutriant, be respectively charged into dialysis in the 3500Da dialysis tubing, concentrate solution freeze-drying in the dialysis tubing.Wherein the white flocks by 1.0mol/L NaCl wash-out gained is the fucoidan-falactosan sulfuric ester.Its structure is identified and is seen embodiment 2.
Embodiment 2: the structure of fucoidan-falactosan sulfuric ester is identified
(1) desulphurization reaction of fucoidan-falactosan sulfuric ester.
Hydrochloric acid-pyridine with 1mol/L carries out pre-treatment to HZ001 type resin earlier, taking by weighing 1.0g fucoidan-falactosan sulfuric ester then is dissolved in the 50mL deionized water, through HZ001 type resin, with 10 column volumes of distillation washing, collect the washing component, concentrate, with pH furnishing neutrality, freeze-drying gets the polysaccharide pyridinium salt with pyridine.The above-mentioned polysaccharide pyridinium salt of 0.5g is dissolved in 100mL methyl-sulphoxide-methyl alcohol-pyridine solution (volume ratio 89: 10: 1), 100 ℃ of reaction 9h.After reaction finishes, with the reaction solution dialysis, freeze-drying, and detect the degree of desulfurization acidic group by sulfate assay and infrared spectra.
(2) methylation analysis of fucoidan-falactosan sulfuric ester
A. agent treated
Get the molecular sieve of 25g, be added to the micro-moisture of removing in the 100mL methyl-sulphoxide in the reagent through 500 ℃ of freeze-day with constant temperature 4h of retort furnace; Leave in after methyl iodide heavily steams CaCl is housed
2Brown bottle in.
B. methylation reaction
Fucoidan-falactosan sulfuric ester sample is put drying under reduced pressure 48h in the phosphorus pentoxide desiccator, and get the dry polysaccharide of 3mg and place the 10mL centrifuge tube, with the dissolving of 1mL methyl-sulphoxide, logical N
2, adding exsiccant NaOH powder 100mg, room temperature concussion 30min drips the 0.8mL methyl iodide in ice bath, room temperature concussion 30min.Add 1mL water termination reaction, use N
2Blow out unreacted methyl iodide, use 1mL chloroform extraction 2 times, wash chloroform layer (2mL*3) with water, chloroform layer is at N
2Volatilize in the stream.Repeat 3-4 hydroxyl peak disappearance in the IR spectrogram.
C.GC/MS analyzes
The polysaccharide that methylates is dissolved in 100 ℃ of hydrolysis 6h with 1mL formic acid, and reaction finishes the evaporate to dryness reaction solution, adds the trifluoroacetic acid of 2mL 2mol/L, 100 ℃ of reaction 6h.With the hydrolyzed solution evaporate to dryness, with methanol wash (3*3mL).Sample is dissolved among 2mL water and the 1mL 0.05mol/L NaOH, adds 25mgNBH
4, 25 ℃ of reaction 2h, with the acetic acid neutralization, methanol wash (4*3mL) is dissolved in 2mL water, freeze-drying behind the evaporate to dryness.Freeze dried sample is dissolved with the 1mL pyridine, and 90 ℃ of reaction 30min add the 1mL diacetyl oxide again, 100 ℃ of reaction 1h, and with the reaction solution evaporate to dryness, with the methylene dichloride dissolving, washing dichloromethane layer (3*2mL).
Chromatographic condition: chromatographic column: Fused Silicon Column 30QC3DB225; Column temperature is since 100 ℃ of intensifications, is incubated 15min after being raised to 220 ℃ with 5 ℃/min speed; 250 ℃ of injector temperatures; Carrier gas: helium; Mass spectrum vacuum tightness is 14.5psi; Ion source: EI70ev; Detector: MSD; Mass scanning scope: 30-500au; Sample size: 1 μ L; Flow velocity: 1.0mL/min.
(3) the NMR Spectrum Analysis of fucoidan-falactosan sulfuric ester and desulfurization fucoidan-falactosan sulfuric ester
Fucoidan-falactosan sulfuric ester and desulfurization fucoidan-falactosan sulfuric ester sample are dissolved with deuterium-oxide, and freeze-drying repeats twice.With 99.97% deuterium-oxide sample being made into the solution of 2-3% again, is interior mark with AVANCE600-MHz nmr determination DDS.
1H NMR and
13C NMR measures under 310K.
(4) experimental result
IR spectrogram such as Fig. 2 of fucoidan-falactosan sulfuric ester and desulfurization product desulfurization fucoidan-falactosan sulfuric ester thereof, sample is at 1250cm after the desulfurization
-1Near strong absorption peak disappear, and this position is the characteristic absorbance of the stretching vibration of S=O, the variation that further feature absorbs is less, illustrates that desulfurization is successful and does not destroy the skeleton structure of polysaccharide.
Fig. 3 is the infrared spectrum after the methylating of fucoidan-falactosan sulfuric ester and desulfurization fucoidan-falactosan sulfuric ester.As can be seen from the figure, at 3700-3100cm
-1The absorption peak place of hydroxyl does not have the peak, originally 3440cm
-1Near strong peak has also disappeared, and illustrates that hydroxyl is modified.At 2960cm
-1And 2922cm
-1Strong peak appears in the place, and is very weak peak originally herein, and these two peaks can belong to the stretching vibration absorption peak of C-H, and these peaks have illustrated the existence of methyl, and proving methylates is completely.The methylation analysis result is as shown in table 3.The NMR Spectrum Analysis result of fucoidan-falactosan sulfuric ester and desulfurization fucoidan-falactosan sulfuric ester is shown in table 2 and 3.
Embodiment 3: the fucoidan-falactosan sulfuric ester is to the therapeutic action of chronic renal failure rat
1, chronic kidney hypofunction Modelling
Adult SPF level Wistar male rat, raise after buying with normal diet and observe a week, after general state is good, take out 10 at random as normal control group (physiological saline 10mL/kg), grouping immediately after all the other 50 animals are weighed, with the administration of 300mg/kg/d VITAMIN B4 oral administration gavage once a day, continuous modeling is 21 days.Modeling finishes the back blood sampling, every rat blood sampling 2-3mL, after the blood sampling back is placed 2 hours in the laboratory, 3000 rev/mins centrifugal 10 minutes, get supernatant, detect blood creatinine and urea nitrogen content, according to experimental result, get modeling after animal be divided into 5 groups immediately, 10 every group.
2. the animal pattern administration is raised
Totally 6 groups of laboratory animal, be respectively: normal control group (normal diet, physiological saline 10mL/kg), model control group (normal diet, physiological saline 10mL/kg), positive controls (extra large elder brother's kidney happiness capsule 220mg/Kg, lot number 20090326, Huinan, Jilin Province long queue Pharma Inc.) group and according to the fucoidan-falactosan sulfuric ester height (100mg/Kg) of the method for embodiment 1 preparation, in (50mg/Kg), low (25mg/Kg) three treatment groups, gastric infusion 28 days.
Leave and take painstaking effort chemical examination blood plasma total protein (TP), albumin (A), blood urea nitrogen (BUN), flesh liver (Scr) before the execution.
3. experimental result
Table 4 fucoidan-falactosan sulfuric ester is to the mensuration of chronic kidney hypofunction rat blood index
| Sample number into spectrum | TP total protein g/L | ALB albumin g/L | Serum urea nitrogen mM/L | Serum creatinine μ M/L |
| Normal group | 75.3±5.5 | 35.1±2.6 | 9.4±0.9 ** | 69.1±3.48 ** |
| Model group | 78.9±7.4 | 30.6±1.9 # | 42.9±10.8 ## | 216.0±38.2 ## |
| 220 mg/kg are organized in sea elder brother's kidney happiness | 76.1±3.6 | 31.3±1.9 | 29.1±4.2 ##** | 154.8±23.9 ##** |
| High dose group | 78.9±5.8 | 32.2±2.7 | 22.9±3.4 ##** | 134.8±13.9 ##** |
| Middle dosage group | 83.6±5.9 # | 34.4±3.9 | 24.3±6.8 ##** | 140.5±26.1 ##** |
| Low dose group | 79.7±5.4 | 34.9±3.2 * | 25.1±4.4 ##** | 139.9±22.6 ##** |
#: compared significant difference with normal group;
##: compared utmost point significant difference with normal group;
*: compare significant difference with model group;
*: compared a grade significant difference with model group
Chronic kidney hypofunction rat due to the oral administration gavage administration VITAMIN B4, with the normal control group relatively, serum creatinine and urea nitrogen levels all are significance and raise (p＜0.01), albumin obviously descend (p＜0.05).Treat after 30 days, fucoidan-falactosan sulfuric ester serum creatinine and urea nitrogen levels significantly reduce (p＜0.01) than model control group, and this effect strengthens along with the increase of dosage, and total serum protein and albumin level are not had influence (table 4) substantially.The result shows that the fucoidan-falactosan sulfuric ester demonstrates chronic kidney hypofunction good curing and kidney provide protection.Chronic kidney hypofunction is because the renal function that causes of multiple disease carrying out property disappearance slowly.Serum creatinine and blood urea nitrogen are two important indicators of disease severity.The fucoidan-falactosan sulfuric ester significantly reduces serum creatinine and urea nitrogen levels, means that the fucoidan-falactosan sulfuric ester might become a kind of medicine of promising treatment chronic kidney hypofunction.
1 test materials
1.1 test drug and reagent
The fucoidan-falactosan sulfuric ester is according to embodiment 1 described method preparation
Sea elder brother's kidney happiness capsule, lot number 20090326, Huinan, Jilin Province long queue Pharma Inc. produces.
Streptozotocin: the Sigma product, lot number: S-0130 faces the time spent and prepares with pH4.4 citric acid damping fluid.
Glurenorm tablet (gliquidone): the 30mg/ sheet, lot number: 070827, the Beijing WanHui ShuangHe pharmacy Co.,Ltd.
Lotensin sheet (benazepril hydrochloride): 10mg/ sheet, lot number: X1009, Novartis Pharma AG.
Reagent kit of glucose: bio-engineering research institute product, lot number: 20070706 are built up in Nanjing.
The urine protein test kit: bio-engineering research institute product, lot number: 20080402 are built up in Nanjing.
Uric creatinine is measured test kit: bio-engineering research institute product, lot number: 20080614 are built up in Nanjing.
Urinary albumin: put and exempt from medicine box, atom High Seience Technology Co., Ltd. product.
Microglobulin is put and is exempted from medicine box: put and exempt from medicine box, atom High Seience Technology Co., Ltd. product.
Regular Insulin: put and exempt from medicine box, atom High Seience Technology Co., Ltd. product.
1.2 test apparatus
TU-1800/1800S ultraviolet-visible spectrophotometer: Beijing Puxi General Instrument Co., Ltd's product.
Automatic clinical chemistry analyzer: daily output, Olympus Au640.
1.3 experimental animal
Wistar kind rat is provided by medical courses in general institute animal, conformity certification number: SCXK capital 2005-0013.
1.4 feeding and management
Feed: the Tianjin animal center provides.
Animal Lab.: room temperature is controlled at 22 ± 2 ℃ by central air-conditioning, and humidity is 50 ± 15%.
2 test methods
2.1 the fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat
Select 130 of healthy male Wistar kind rats for use, body weight 200-230g, vetanarcol 40mg/kg intraperitoneal anesthesia, sterilization is after skin is cut at the back, separating muscle, complete excision right side kidney (10 sham operated rats only undergo surgery operation, do not extract the right side kidney), suture muscles and skin then, after conventional 2 weeks of raising, with streptozotocin 50mg/kg abdominal injection, after 48 hours, measure fasting plasma glucose and glucose in urine.The rat of meet the requirements (fasting plasma glucose 17.1～31.4mmol/L and glucose in urine strong positive) at random equilibrium be divided into model control group, fucoidan-falactosan sulfuric ester high dose group (100mg/kg), middle dosage group (50mg/kg), low dose group (25mg/kg), positive drug control group (Glurenor 15mg/kg+ benazepril 1.5mg/kg), every group is 12, add sham operated rats, totally 6 groups are carried out gastric infusion, irritate the stomach volume and be the 1ml/100g body weight, model control group, Sham-operated control group is all irritated stomach and is waited capacity 0.5%CMC, once a day, 7 times weekly, in continuous 10 weeks, observe and measure following index.
(1) overview
Observe the mental status of animal, diet, hair color, body weight etc.
(2) twenty-four-hour urine protein quantification
Water collection rat twenty-four-hour urine liquid is can't help in fasting in the rat metabolic cage, builds up the test kit operation instructions mensuration urine protein that bio-engineering research is produced by Nanjing.
(3) blood sugar
After the fasting 4 hours, etherization, kapillary eye socket are got the about 50 μ l of blood, centrifugal after, build up the test kit operation instructions that bio-engineering research produces by Nanjing and measure blood sugar.
(4) blood, urine biochemistry
Off-test behind the collection rat twenty-four-hour urine liquid, is built up the test kit operation instructions mensuration uric creatinine that bio-engineering research is produced with Nanjing; Abdominal aortic blood is measured serum creatinine, blood urea nitrogen with Olympus Au640 automatic clinical chemistry analyzer, and calculates endogenous creatinine clearance rate.
(5) trace ingredients
Off-test is measured microalbumin, microglobulin, serum insulin and saccharification hemoglobin content with putting the method for exempting from.
(6) kidney organ coefficient
Weighing rat limosis body weight and kidney are heavy, are the kidney coefficient with the kidney recast of 100g body weight rat.
(7) renal tissue pathological examination
Off-test, kidney 10% formalin fixed, the pathological change of kidney is observed in HE dyeing under opticmicroscope, and calculates renal glomerulus diameter and area.
3 test-results
3.1 overview
The sham operated rats rat body weight increases obviously, and the mental status is good, moves freely, and hair color is normal; The model group rat obviously becomes thin, and is slow in reacting, perpendicular hair, and cataractous eyeball occurring is 7/20; The fucoidan-falactosan sulfuric ester 100 and the 50mg/kg dosage group mental status are better than model control group, less perpendicular hair, and reaction better cataractous eyeball occurs and is respectively 3/20,4/16; 165mg/kg dosage group and model control group compare, no significant difference, and cataractous eyeball occurring is 5/18, and positive drug (lotensin+Glurenor) is organized also significantly better than model control group, and cataractous eyeball occurring is 4/20; Compare with model group, each dosage group body weight is not seen notable difference, the results are shown in Table 5.
3.2 twenty-four-hour urine amount, urine protein are quantitative
Compare with sham operated rats, in 2,4,6,8,10 weeks after the administration, model group rat twenty-four-hour urine amount, urine protein quantitatively all obviously increase; With model group relatively, fucoidan-falactosan sulfuric ester 100,50mg/kg dosage group all obviously reduce by 6,8,10 all twenty-four-hour urine amounts and twenty-four-hour urine protein quantifications after the administration, the results are shown in Table 6, table 7.
Table 5 fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat body weight
(n)
▲: P＜0.01 (comparing) n: number of animals with model group
Table 6 fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat twenty-four-hour urine amount
(n)
△ △: P＜0.01 (comparing) with sham operated rats
*: P＜0.05
*: P＜0.01 (comparing) n: number of animals with model group.
Table 7 fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat twenty-four-hour urine protein content
(n)
△ △: P＜0.01 (comparing) with sham operated rats
*: P＜0.05
*: P＜0.01 (comparing) n: number of animals with model group
3.3 blood sugar
Compare with sham operated rats, in 0,2,4,6,10 weeks after the administration, the model group rat blood sugar obviously raises; Compare with model control group, fucoidan-falactosan sulfuric ester 100mg/kg group the 8th, 10 weeks after administration are obviously reduced rat blood sugar, positive drug (Glurenor+lotensin) is organized after administration and the 4th, 6,8,10 weeks was obviously reduced rat blood sugar, other administration group does not all have obvious influence to rat blood sugar, the results are shown in Table 8.
Table 8 fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat blood sugar
(n)
▲: P＜0.01 (comparing) with sham operated rats
*: P＜0.05
*: P＜0.01 (comparing) with model group; N: number of animals
3.4 biochemical indicator
Compare with sham operated rats, model group serum creatinine, serum urea nitrogen obviously raise, and each administration group does not all have obvious influence to serum creatinine, and fucoidan-falactosan sulfuric ester 50,100mg/kg dosage group all have obvious reduction effect to serum urea nitrogen.Fucoidan-falactosan sulfuric ester 100mg/kg and Glurenor+lotensin dosage group CrCl obviously raises.The results are shown in Table 9.
Table 9 fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat serum creatinine, blood urea nitrogen
△ △: P＜0.01 (comparing) with sham operated rats
*: P＜0.05
*P＜0.01 (comparing) with model group
3.5 trace ingredients
Compare with sham operated rats, the content of model group rat blood serum Regular Insulin, glycolated hemoglobin, β2Wei Qiudanbai obviously reduces; Compare with model control group, fucoidan-falactosan sulfuric ester 100mg/kg obviously increases the rat microalbumin, fucoidan-falactosan sulfuric ester 25mg/kg rising β2Wei Qiudanbai, and high, middle dosage also has the trend of increase, and Glurenor+lotensin also has similar action.Each administration group does not all have obvious influence to other every indexs, sees Table 10.
Table 10 fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat trace ingredients
△: P＜0.05
△ △: P＜0.01 (comparing) with sham operated rats
*: P＜0.05
*: P＜0.01 (comparing) with model group
3.6 kidney coefficient:
Compare with sham operated rats, model group rat kidney hypertrophy, organ coefficient obviously increases; Compare with model control group, fucoidan-falactosan sulfuric ester 50,100mg/kg and Glurenor+lotensin dosage group kidney organ coefficient obviously reduces, and sees Table 11.
Table 11 fucoidan-falactosan sulfuric ester is to the influence of diabetic nephropathy rat kidney organ coefficient
△ △: P＜0.01 (comparing) with sham operated rats
*: P＜0.05 (comparing) with model group
3.7 kidney pathology
The result shows that sham operated rats renal tissue form is not seen change, and the model group renal lesions is obvious, glomerular volume increases, and cell count increases, the expansion of part glomerular capillary, renal cells vacuolar degeneration, each dosage group renal lesions all have alleviating in various degree; Compare with model group, fucoidan-falactosan sulfuric ester 50,100mg/kg dosage group obviously reduce glomerular capsule girth, sectional area and cell count.
4 experiment conclusion
Experimental result shows that the fucoidan-falactosan sulfuric ester has tangible preventive and therapeutic effect to the rat diabetes ephrosis.
Experimental technique:
Get 70 of SD rats, male, body weight 200-220g is divided into 7 groups at random and is respectively blank group, model group control group, positive control I group (0.1mg/kg dexamethasone acetate), positive control II group (220mg/kg sea elder brother's kidney happiness capsule), fucoidan-falactosan sulfuric ester high dose group (100mg/kg), middle dosage group (50mg/kg), low dose group (25mg/kg).Oral successive administration 3d, be administered twice every day, morning and afternoon respectively once, dosage is 10ml/kg, in administration beginning in the 4th day modeling, all animals is prohibited water 16h before the modeling, and all the other each treated animals give 50% glycerine physiological saline 10ml/kg except that the blank group, in the injection of rat both sides hindlimb muscle, blank group injection equivalent physiological saline.Successive administration 3d again after the modeling, be administered twice every day, morning and afternoon each once, dosage is 10ml/kg, administration finishes all rat fasting of back, gets serum and detects creatinine, blood urea nitrogen, total protein, albumin respectively, gets anticoagulation and detects red corpuscle, oxyphorase.
Statistical procedures: all data are used
To carrying out the Student`s check between each group, adopt Sun Ruiyuan, Chen Zhiyang, chief editors' such as professor Zheng Qingshan DAS software carries out statistical procedures to The above results, judges significant difference.
Test-results
3.1.1 influence to blood biochemical
The result shows, in the rat acute renal failure model that glycerine causes, model control group and blank group are relatively, serum creatinine, blood urea nitrogen significantly raise (P＜0.05), serum albumin and total protein decrease to a certain extent, illustrate by this test to set up rat acute renal failure model.The fucoidan-falactosan sulfuric ester was with the dosage successive administration of 100mg/kg, 50mg/kg, 25mg/kg 7 days, administration every day can be alleviated the generation of animal renal failure symptom for 2 times, high dosage and middle dosage group have significantly reduced the content of serum creatinine, with model control group significant difference (P＜0.05) are arranged relatively; Each dosage group all can reduce the content (P＜0.05) of serum urea nitrogen; Content and model control group that middle dosage can significantly reduce serum calcium relatively have significant difference (P＜0.01); Each dosage group is to the not significantly influence of content of serum paraoxonase.Compare with extra large elder brother's kidney happiness capsule of Isodose, the fucoidan-falactosan sulfuric ester is better than extra large elder brother's kidney happiness at the action effect that reduces serum creatinine and blood urea nitrogen.The results are shown in Table 12,13.
The influence of rat acute renal failure model creatinine and blood urea nitrogen due to the table 12 pair glycerine (
N=10)
Compare * P＜0.05, * * P＜0.01 with model control group
Compare △ P＜0.05, △ △ P＜0.01 (as follows) with the blank group
Table 13 fucoidan-falactosan sulfuric ester to the influence of rat acute renal failure model calcium due to the glycerine and phosphorus (
N=10)
3.1.2 to hematological influence
The result shows, in the rat acute renal failure model due to the glycerine, do not cause that rat produces tangible hematology and changes, to the influence useless of the content of RBC number and oxyphorase, model and blank group relatively do not have significant difference, each dosage group of fucoidan-falactosan sulfuric ester and model group relatively do not have significant difference (P＞.05), illustrate in the process of being tried the administration of thing acute renal failure short-term can not cause erythrocytic variation.The results are shown in Table 14.
The influence of rat acute renal failure model red corpuscle and oxyphorase due to the table 14 pair glycerine (
N=10)
Conclusion: from above interpretation of result as can be seen the fucoidan-falactosan sulfuric ester acute kidney of rats failure model due to the glycerine is had good protective action.
Claims (6)
1. fucoidan-falactosan sulfuric ester is characterized in that:
It mainly contains Fucose and semi-lactosi; (the 1-residue forms main chain to the sugar chain chemical structure with-4-α-L-Fuc-(1 → 3)-α-L-Fuc-(1 → 3)-α-L-Fuc-(1 → 3)-α-L-Fuc-, 1 → 3 Fucose accounts for 75% in the main chain, 1 → 4 Fucose accounts for 25%, average per 4 saccharide residues contain a side chain, and (1-or β-D-Gla-(1 → 6)-(1-forms β-D-Gla-side chain by α-L-Fuc; Sulfate is connected on 2,4 of Fucose or semi-lactosi 3,4.
2. according to the described fucoidan-falactosan sulfuric ester of claim 1, it is characterized in that: it comprises two structural unit structure A and structure B; Structure A constitutes main chain by 1 → 3 Fucose and 1 → 4 Fucose, is connected with α-L-Fuc (1-side chain on 2 of the Fucose 1 → 3; Structure B constitutes main chain by 1 → 3 Fucose and 1 → 4 Fucose, is connected with β-D-Gla-(1 → 6)-β-D-Gla-(1-side chain on 2 of the Fucose 1 → 3;
3. according to the described fucoidan-falactosan sulfuric ester of claim 1, it is characterized in that: it is that AAB structure by 2 structure A and 1 structure B alternately connects and composes main chain successively, and per 12 fucose units constitute a repeating unit; On the 2nd of main chain with the 6th fucosyl residues 2, be connected with the Fucose side chain that 1-is connected, on 4 of the 10th fucosyl residues of main chain, be connected with 2 galactose units that (1 → 6) connects.
4. the extraction separation purification process of the described fucoidan-falactosan sulfuric ester of claim 1, it is characterized in that: operation steps is as follows:
(1) brown alga is removed silt, add water, 60-120 ℃ of heating extracted 1-4 time; United extraction liquid, suction filtration, concentrating under reduced pressure is used ethanol sedimentation, the dry crude extract that gets;
The described amount that adds water is a 10-40 times of frond weight; Described suction filtration helps filter with diatomite; The final volume concentration of described precipitation process ethanol in system is 50-80%;
(2) crude extract is added water and redissolve, making its mass concentration is 1-1.5%, adds magnesium chloride and ethanolic soln, the centrifugal precipitation of removing, collect supernatant liquor, the final concentration of wherein said magnesium chloride in the redissolution thing is 0.05mol/L, and ethanol final weight concentration in system is 20%;
Supernatant liquor adds ethanol sedimentation again through concentrating under reduced pressure, and the final volume concentration of ethanol in system is 50-80%, filters to such an extent that precipitate;
(3) will precipitate water-solublely, and be the dialysis of 3500Da dialysis tubing with molecular weight cut-off, and after dialysis finishes solution decompression in the bag be concentrated, lyophilize gets the algal polysaccharide of purifying;
(4) algal polysaccharide that (3) are obtained is water-soluble, making its mass concentration is 1-3%, with DEAE-Sepharose-CL-6B is the carrier column chromatography, use 0.1mol/L NaCl, 0.5mol/LNaCl, 1.0mol/L NaCl, 1.5mol/L NaCl and 2.0mol/L NaCl eluant solution successively, collect each elutriant, respectively dialysis, freeze-drying; Wherein the white sample by 1.0mol/L NaCl wash-out gained is the fucoidan-falactosan sulfuric ester.
5. the application of the described fucoidan-falactosan sulfuric ester of claim 1 in preparation treatment kidney disease medicine.
6. according to the application of the described fucoidan-falactosan sulfuric ester of claim 5, it is characterized in that: described kidney disease medicine is treatment renal failure, nephrotic syndrome and/or diabetic nephropathy drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010157935 CN102234336B (en) | 2010-04-24 | 2010-04-24 | Fucoidan-galactosan sulfate, extracting, separating, and purifying method thereof, and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010157935 CN102234336B (en) | 2010-04-24 | 2010-04-24 | Fucoidan-galactosan sulfate, extracting, separating, and purifying method thereof, and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102234336A true CN102234336A (en) | 2011-11-09 |
| CN102234336B CN102234336B (en) | 2013-12-25 |
Family
ID=44885504
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010157935 Active CN102234336B (en) | 2010-04-24 | 2010-04-24 | Fucoidan-galactosan sulfate, extracting, separating, and purifying method thereof, and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102234336B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288978A (en) * | 2013-06-08 | 2013-09-11 | 中国海洋大学 | Fucosan sulphate and preparation method and application thereof in preparation of antidiabetic alpha-glycosidase inhibitor |
| CN103467625A (en) * | 2013-09-10 | 2013-12-25 | 南京海兰琪生物科技有限公司 | Method for preparing agar galactan sulfate |
| CN103804504A (en) * | 2014-01-22 | 2014-05-21 | 山东大学（威海） | Low-molecular-weight fucoidan and effect thereof on diabetic nephropathy |
| CN106749439A (en) * | 2016-12-30 | 2017-05-31 | 山东省科学院生物研究所 | New fucoidan oligosaccharide and preparation method and application |
| CN107987179A (en) * | 2017-12-26 | 2018-05-04 | 中国科学院海洋研究所 | A kind of application of low sulphated fucogalactan in immunopotentiator is prepared |
| CN111875714A (en) * | 2020-08-05 | 2020-11-03 | 青岛海洋生物医药研究院股份有限公司 | Low molecular weight sulfated galactan, and preparation method and application thereof |
| CN116726028A (en) * | 2022-04-26 | 2023-09-12 | 北京浩鼎瑞生物科技有限公司 | Use of fucose in IgA nephropathy treatment |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1670028A (en) * | 2004-12-31 | 2005-09-21 | 中国科学院海洋研究所 | Method for preparing L-fucoidan in sea tangle |
-
2010
- 2010-04-24 CN CN 201010157935 patent/CN102234336B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1670028A (en) * | 2004-12-31 | 2005-09-21 | 中国科学院海洋研究所 | Method for preparing L-fucoidan in sea tangle |
Non-Patent Citations (1)
| Title |
|---|
| 《Carbohydrate Research》 20040225 Maria I. Bilan et al. A highly regular fraction of a fucoidan from the brown seaweed Fucus distichus L. 511-517 1-6 第339卷, 第3期 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288978A (en) * | 2013-06-08 | 2013-09-11 | 中国海洋大学 | Fucosan sulphate and preparation method and application thereof in preparation of antidiabetic alpha-glycosidase inhibitor |
| CN103288978B (en) * | 2013-06-08 | 2015-10-28 | 中国海洋大学 | Fucoidan and preparation method thereof and the application in the antidiabetic alpha-glucosidase inhibitor of preparation |
| CN103467625A (en) * | 2013-09-10 | 2013-12-25 | 南京海兰琪生物科技有限公司 | Method for preparing agar galactan sulfate |
| CN103467625B (en) * | 2013-09-10 | 2015-08-19 | 南京海兰琪生物科技有限公司 | A kind of preparation method of agar-agar Polygalactan sulfuric ester |
| CN103804504A (en) * | 2014-01-22 | 2014-05-21 | 山东大学（威海） | Low-molecular-weight fucoidan and effect thereof on diabetic nephropathy |
| CN106749439A (en) * | 2016-12-30 | 2017-05-31 | 山东省科学院生物研究所 | New fucoidan oligosaccharide and preparation method and application |
| CN106749439B (en) * | 2016-12-30 | 2019-09-17 | 山东省科学院生物研究所 | Fucoidan oligosaccharide and the preparation method and application thereof |
| CN107987179A (en) * | 2017-12-26 | 2018-05-04 | 中国科学院海洋研究所 | A kind of application of low sulphated fucogalactan in immunopotentiator is prepared |
| CN107987179B (en) * | 2017-12-26 | 2020-07-07 | 中国科学院海洋研究所 | Application of low-sulfated fucan in preparation of immunopotentiator |
| CN111875714A (en) * | 2020-08-05 | 2020-11-03 | 青岛海洋生物医药研究院股份有限公司 | Low molecular weight sulfated galactan, and preparation method and application thereof |
| CN116726028A (en) * | 2022-04-26 | 2023-09-12 | 北京浩鼎瑞生物科技有限公司 | Use of fucose in IgA nephropathy treatment |
| CN116726028B (en) * | 2022-04-26 | 2023-12-19 | 北京浩鼎瑞生物科技有限公司 | Use of fucose in IgA nephropathy treatment |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102234336B (en) | 2013-12-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102234336B (en) | Fucoidan-galactosan sulfate, extracting, separating, and purifying method thereof, and application thereof | |
| CN101301310B (en) | Use of brown alga polysaccharide sulfate in preventing and treating Parkinson's disease | |
| CN102617745B (en) | Preparation method and blood sugar lowering function of Ganoderma lucidum karst polysaccharide F31 | |
| CN103880910B (en) | A kind of preparation method and its usage of Cyclosiversigenin | |
| KR100361187B1 (en) | The hematopoietic, myeloid protecting, antitumor immune cells generating and radiosensitizing polysaccharide isolated from Panax ginseng | |
| CN108047343B (en) | Preparation method and application of fritillaria pallidiflora total polysaccharide | |
| CN1985846A (en) | Use of brown algae polyose sulfate in preparing medicine fortreating cardic and cerebral vascular diseases | |
| CN103724445A (en) | Preparation method and blood sugar lowering function of Grifola frondosa polysaccharide F2 | |
| CN107722131B (en) | Total ganoderma lucidum spore powder refined polysaccharide with significant auxiliary antitumor activity and preparation method and application thereof | |
| CN110551230A (en) | Preparation method of astragalus polysaccharide | |
| CN101028282B (en) | Use of low-molecular weight phaeophytin polyose sulfate in preparation of medicine for treating kidney disease | |
| CN110790848A (en) | Preparation method and application of total polysaccharides of sea buckthorn | |
| CN106084087A (en) | A kind of preparation method of Fructus Trichosanthis polysaccharide | |
| CN100369936C (en) | Sulfonated changium root polysaccharides and preparing method and use thereof | |
| CN101654485A (en) | Spreading hedvotis herb polysaccharide for curing malignant tumor and preparation method thereof | |
| CN103382229B (en) | A kind of preparation method and Structural Identification with the novel SEP-1 of immunoregulation effect | |
| CN102161710A (en) | Method for preparing tremellan with low molecular weight and novel medicinal application thereof | |
| CN105902562A (en) | Application of glucomannan in preparation of leucocytopenia prevention and treatment drugs | |
| CN102276754B (en) | Organosulfate glucan in hedysarum polybotys saccharide as well as preparation method and application thereof | |
| CN101306014B (en) | Use of brown algae polysaccharide sulfuric acid ester of low molecular weight in preparing medicine for treating diabetic nephropathy | |
| CN101011412A (en) | Usage of low-molecular-weight algal polysaccharide sulfate in preparation of medicament for treating hepatic disease | |
| CN113717296B (en) | Eucommia acidic polysaccharide, extraction method and application of eucommia acidic polysaccharide in preparation of anti-colon cancer drugs | |
| CN107987179B (en) | Application of low-sulfated fucan in preparation of immunopotentiator | |
| CN109369816A (en) | The extracting method of sargassum fusifome anti-aging polysaccharide | |
| CN108948223B (en) | Myrtle polysaccharide P1, its separation method and application in preparing hypolipidemic medicine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20111109 Assignee: Huinan Changlong Biochemical Pharmaceutical Co., Ltd., Jilin Prov. Assignor: Institute of Oceanology, Chinese Academy of Sciences Contract record no.: 2014220000051 Denomination of invention: Fucoidan-galactosan sulfate, extracting, separating, and purifying method thereof, and application thereof Granted publication date: 20131225 License type: Exclusive License Record date: 20141218 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model |